Julie A. Champion

Making polymeric micro- and nanoparticles of complex shapes.

Polymeric micro- and nanoparticles play a central role in varied applications such as drug delivery, medical imaging, and advanced materials, as well as in fundamental studies in fields such as microfluidics and nanotechnology. Functional behavior of polymeric particles in these fields is strongly influenced by their shape. However, the availability of precisely shaped polymeric particles has been a major bottleneck in understanding and capitalizing on the role of shape in particle function. Here we report a method that directly addresses this need. Our method uses routine laboratory chemicals and equipment to make particles with >20 distinct shapes and characteristic features ranging in size from 60 nm to 30 μm. This method offers independent control over important particle properties such as size and shape, which is crucial to the development of nonspherical particles both as tools and products for a variety of fields.

Control of endothelial targeting and intracellular delivery of therapeutic enzymes by modulating the size and shape of ICAM-1-targeted carriers.

Endocytosis in endothelial cells (ECs) is important for many biomedical applications, including drug delivery by nano- and microscale carriers. However, little is known about how carrier geometry influences endothelial drug targeting, intracellular trafficking, and effects. We studied this using prototype polymer carriers of various sizes (0.1-10 mum) and shapes (spheres versus elliptical disks). Carriers were targeted to intercellular adhesion molecule 1 (ICAM-1), a transmembrane glycoprotein that is upregulated in many pathologies and used as a target for intraendothelial drug delivery. ECs internalized anti-ICAM-coated carriers of up to several microns in size via cell adhesion molecule-mediated endocytosis. This pathway is distinct from caveolar and clathrin endocytosis that operate for submicron-size objects. Carrier geometry was found to influence endothelial targeting in the vasculature, and the rate of endocytosis and lysosomal transport within ECs. Disks had longer half-lives in circulation and higher targeting specificity in mice, whereas spheres were endocytosed more rapidly. Micron-size carriers had prolonged residency in prelysosomal compartments, beneficial for endothelial antioxidant protection by delivered catalase. Submicron carriers trafficked to lysosomes more readily, optimizing effects of acid sphingomyelinase (ASM) enzyme replacement in a model of lysosomal storage disease. Therefore, rational design of carrier geometry will help optimize endothelium-targeted therapeutics.

Shape induced inhibition of phagocytosis of polymer particles.

PurposeTo determine if particle shape can be engineered to inhibit phagocytosis of drug delivery particles by macrophages, which can be a significant barrier to successful therapeutic delivery.MethodsNon-spherical polystyrene particles were fabricated by stretching spherical particles embedded in a polymer film. A rat alveolar macrophage cell line was used as model macrophages. Phagocytosis of particles was assessed using time-lapse video microscopy and fluorescence microscopy.ResultsWe fabricated worm-like particles with very high aspect ratios (>20). This shape exhibits negligible phagocytosis compared to conventional spherical particles of equal volume. Reduced phagocytosis is a result of decreasing high curvature regions of the particle to two single points, the ends of the worm-like particles. Internalization is possible only at these points, while attachment anywhere along the length of the particles inhibits internalization due to the low curvature.ConclusionsShape-induced inhibition of phagocytosis of drug delivery particles is possible by minimizing the size-normalized curvature of particles. We have created a high aspect ratio shape that exhibits negligible uptake by macrophages.

Cell-selective metabolic labeling of proteins

Metabolic labeling of proteins with the methionine surrogate azidonorleucine can be targeted exclusively to specified cells through expression of a mutant methionyl-tRNA synthetase (MetRS). In complex cellular mixtures, proteins made in cells that express the mutant synthetase can be tagged with affinity reagents (for detection or enrichment) or fluorescent dyes (for imaging). Proteins made in cells that do not express the mutant synthetase are neither labeled nor detected.

Protein Nanoparticles for Intracellular Delivery of Therapeutic Enzymes

The use of enzymes as therapeutics is very promising because of their catalytic activity and specificity. However, intracellular delivery of active enzymes is challenging due to their low stability and large size. The production of protein-enzyme nanoparticles was investigated with the goal of developing a protein carrier for active enzyme delivery. β-Galactosidase (β-gal), an enzyme whose deficiency is the cause of some lysosomal storage disorders, was incorporated into enhanced green fluorescent protein nanoparticles prepared via desolvation. Particle size was found to be sensitive to the type of cross-linker, cross-linking time, and the presence of imidazole. The results indicate that β-gal activity is highly retained (>70%) after particle fabrication and >85% of protein is incorporated in the particles. Protein-enzyme nanoparticles exhibited higher internalization in multiple cell lines in vitro, compared with the soluble enzyme. Importantly, β-gal retained its activity following intracellular delivery. These data demonstrate that protein nanoparticles are a biocompatible, high-efficiency alternative for intracellular delivery of active enzyme therapeutics.

Nanoclusters self-assembled from conformation-stabilized influenza M2e as broadly cross-protective influenza vaccines

UNLABELLED Influenza vaccines with broad cross-protection are urgently needed. The highly conserved ectodomain of the influenza matrix protein 2 (M2e) can be a promising candidate if its low immunogenicity was overcome. In this study, we generated protein nanoclusters self-assembled from conformation-stabilized M2e tetramers (tM2e) to improve its immunogenicity. The resulting nanoclusters showed an average hydrodynamic diameter of 227 nm. Vaccination with the nanoclusters by an intranasal route elicited high levels of serum antigen-specific IgG in mice (approximately 100-fold higher than that obtained with soluble tM2e), as well as antigen-specific T cell and mucosal antibody responses. The immunity conferred complete protection against lethal challenge with homo- as well as heterosubtypic viruses. These results demonstrate that nanoclusters assembled from conformation-stabilized M2e are promising as a potential universal influenza A vaccine. Self-assembly into nanoclusters represents a novel approach for increasing the immunogenicity of vaccine antigens. FROM THE CLINICAL EDITOR In order to develop more effective influenza vaccination, the highly conserved ectodomain of M2e could be a promising candidate. Unfortunately, it is a weak antigen for vaccination purposes. In this study, self-assembled protein nanoclusters of tM2e were generated and tested. The nanoclusters demonstrated superior vaccination properties, with complete protection against lethal challenge in the studied rodent model, raising hope for the introduction of similar vaccines to challenge human influenza outbreaks.

State-Selective Metabolic Labeling of Cellular Proteins

Transcriptional activity from a specified promoter can provide a useful marker for the physiological state of a cell. Here we introduce a method for selective tagging of proteins made in cells in which specified promoters are active. Tagged proteins can be modified with affinity reagents for enrichment or with fluorescent dyes for visualization. The method allows state-selective analysis of the proteome, whereby proteins synthesized in predetermined physiological states can be identified. The approach is demonstrated by proteome-wide labeling of bacterial proteins upon activation of the P(BAD) promoter and the SoxRS regulon and provides a basis for analysis of more complex systems including spatially heterogeneous microbial cultures and biofilms.

Double-layered protein nanoparticles induce broad protection against divergent influenza A viruses

Current influenza vaccines provide limited protection against circulating influenza A viruses. A universal influenza vaccine will eliminate the intrinsic limitations of the seasonal flu vaccines. Here we report methodology to generate double-layered protein nanoparticles as a universal influenza vaccine. Layered nanoparticles are fabricated by desolvating tetrameric M2e into protein nanoparticle cores and coating these cores by crosslinking headless HAs. Representative headless HAs of two HA phylogenetic groups are constructed and purified. Vaccinations with the resulting protein nanoparticles in mice induces robust long-lasting immunity, fully protecting the mice against challenges by divergent influenza A viruses of the same group or both groups. The results demonstrate the importance of incorporating both structure-stabilized HA stalk domains and M2e into a universal influenza vaccine to improve its protective potency and breadth. These potent disassemblable protein nanoparticles indicate a wide application in protein drug delivery and controlled release.Relatively well conserved domains of influenza A virus (IAV) proteins are potential candidates for the development of a universal IAV vaccine. Here, Deng et al. combine two such conserved antigens (M2e and HA stalk) in a double-layered protein nanoparticle and show that it protects against divergent IAVs in mice.

Protein nanoparticle vaccine based on flagellin carrier fused to influenza conserved epitopes confers full protection against influenza A virus challenge

Currently marketed influenza vaccines only confer protection against matching influenza virus strains. The influenza A composition of these vaccines needs to be annually updated. Vaccines that target conserved epitopes of influenza viruses would in principle offer broad cross-protection against influenza A viruses. In our study, we investigated the specific immune responses and protective efficacy of protein nanoparticles based on fusion proteins of flagellin carrier linked to conserved influenza epitopes. We designed fusion proteins by replacing the hyperimmunogenic region of flagellin (FliC) with four tandem copies of the ectodomain of matrix protein 2 (f4M2e), H1 HA2 domain (fHApr8) or H3 HA2 domain (fHAaichi). Protein nanoparticles fabricated from these fusion proteins by using DTSSP crosslinking retained Toll-like receptor 5 agonist activity of FliC. Intranasal immunization with f4M2e, f4M2e/fHApr8 or f4M2e/fHAaichi nanoparticles induced vaccine antigen-specific humoral immune responses. It was also found that the incorporation of the H1 HA2 domain into f4M2e/fHApr8 nanoparticles boosted M2e specific antibody responses. Immunized mice were fully protected against lethal doses of virus challenge.

Engineering Polyelectrolyte Capsules with Independently Controlled Size and Shape

Polyelectrolyte capsules (PECs) are a promising delivery system that has the ability to carry a large payload of a variety of cargoes. Controlling PEC properties is critical to understanding and tuning their cellular uptake efficiency, kinetics, and mechanism as well as their biodistribution in the body. The lack of a method to independently engineer PEC size, shape, and chemistry impedes both basic understanding of how physicochemical parameters affect PEC behavior in drug delivery and other applications, and the ability to optimize parameters for best function. Here, we report the successful fabrication of PECs having constant surface chemistry with independently controlled size and shape by combining soft organic templates created by the particle stretching method and a modified layer-by-layer (LBL) deposition process. Changing the template dispersion solution during LBL deposition from water to ethanol allowed us to overcome previous issues with organic templates, such as aggregation and template removal. These results will contribute not only to the basic study of the role of capsule shape and size on its function but also to the optimization of capsule properties for drug or imaging carriers, sensors, reactors, and other applications.